Naturalized Escherichia coli in Wastewater and the Co-evolution of Bacterial Resistance to Water Treatment and Antibiotics.

Antibiotic resistance represents one of the most pressing concerns facing public health today. While the current antibiotic resistance crisis has been driven primarily by the anthropogenic overuse of antibiotics in human and animal health, recent efforts have revealed several important environmental dimensions underlying this public health issue. Antibiotic resistant (AR) microbes, AR genes, and antibiotics have all been found widespread in natural environments, reflecting the ancient origins of this phenomenon. In addition, modern societal advancements in sanitation engineering (i.e., sewage treatment) have also contributed to the dissemination of resistance, and concerningly, may also be promoting the evolution of resistance to water treatment. This is reflected in the recent characterization of naturalized wastewater strains of Escherichia coli-strains that appear to be adapted to live in wastewater (and meat packing plants). These strains carry a plethora of stress-resistance genes against common treatment processes, such as chlorination, heat, UV light, and advanced oxidation, mechanisms which potentially facilitate their survival during sewage treatment. These strains also carry an abundance of common antibiotic resistance genes, and evidence suggests that resistance to some antibiotics is linked to resistance to treatment (e.g., tetracycline resistance and chlorine resistance). As such, these naturalized E. coli populations may be co-evolving resistance against both antibiotics and water treatment. Recently, extraintestinal pathogenic strains of E. coli (ExPEC) have also been shown to exhibit phenotypic resistance to water treatment, seemingly associated with the presence of various shared genetic elements with naturalized wastewater E. coli. Consequently, some pathogenic microbes may also be evolving resistance to the two most important public health interventions for controlling infectious disease in modern society-antibiotic therapy and water treatment.

Characterization of water treatment-resistant and multidrug-resistant urinary pathogenic Escherichia coli in treated wastewater.

A growing body of evidence has demonstrated that extraintestinal pathogenic E. coli (ExPEC), such as the urinary pathogenic E. coli (UPEC), are common constituents of treated wastewater, and therefore represent a potential public health risk. However, no single virulence gene, or set of virulence genes, can be used to conclusively identify this genetically diverse pathotype. As such we sought to identify and characterize the public health relevance of potential UPEC found in treated sewage/wastewater using a comparative genomics approach. Presumptive wastewater UPEC (W-UPEC) were initially identified by virulence gene screening against 5 virulence genes, and for which isolates containing ≥3 virulence genes were whole genome sequenced (n = 24). Single nucleotide polymorphic (SNP) spanning tree analysis demonstrated that many of these wastewater UPEC (WUPEC) were virtually identical at the core genome (0.4 Mbp) when compared to clinical UPEC (C-UPEC) sequences obtained from NCBI, varying by as little as 1 SNP. Remarkably, at the whole genome level, W-UPEC isolates displayed >96% whole genome similarity to C-UPEC counterparts in NCBI, with one strain demonstrating 99.5% genome similarity to a particular C-UPEC strain. The W-UPEC populations were represented by sequence types (ST) known to be clinically important, including ST131, ST95, ST127 and ST640. Many of the W-UPEC carried the exact same complement of virulence genes as their most closely related C-UPEC strains. For example, O25b-ST131 W-UPEC strains possessed the same 80 virulence genes as their most closely related C-UPEC counterparts. Concerningly, W-UPEC strains also carried a plethora of antibiotic resistance genes, and O25b-ST131strains were designated as extended spectrum beta-lactamase (ESBL) producing E. coli by both genome profiling and phenotypic resistance testing. W-UPEC ST131 strains were found in the effluents of a single treatment plant at different times, as well as different wastewater treatment plants, suggesting a differentially ability to survive wastewater treatment. Indeed, in sewage samples treated with chlorine doses sufficient for inducing a ∼99.99% reduction in total E. coli levels, UPEC represented a significant proportion of the chlorine-resistant population. By contrast, no Shiga toxin-producing E. coli were observed in these chlorinated sewage libraries. Our results suggest that clinically-relevant UPEC exist in treated wastewater effluents and that they appear to be specifically adapted to survive wastewater treatment processes.

Survival, prophage induction, and invasive properties of lysogenic Salmonella Typhimurium exposed to simulated gastrointestinal conditions.

This study was designed to evaluate the viability, prophage induction, invasive ability, and relative gene expression in lysogenic Salmonella Typhimurium exposed to the simulated gastric juice (SGJ) at pH 2 (SGJ-2), 3 (SGJ-3), 4 (SGJ-4), and 5 (SGJ-5) for 30 min followed by 0.5 % bile salts for 2 h. The susceptibility of lysogenic S. Typhimurium increased with decreasing pH value and increasing bile salt concentration. The lysogenic S. Typhimurium cells were least susceptible to SGJ-4 and SGJ-5, showing <1 log reduction. The highest prophage induction was observed by 3.34 log PFU/ml in lysogenic S. Typhimurium at SGJ-3 in the presence of 0.5 % bile salts. The numbers of invading lysogenic S. Typhimurium treated at SGJ-3, SGJ-4, and SGJ-5 were 3.57, 3.73, and 4.15 log CFU/cm(2), respectively. Most genes (hilA, hilC, hilD, invA, invE, invF, and sirA) were down-regulated in lysogenic S. Typhimurium treated at SGJ-3, SGJ-4, and SGJ-5. This study provides useful information for understanding physiological changes of lysogenic S. Typhimurium in the simulated gastrointestinal conditions.

Effect of bacteriophage on the susceptibility, motility, invasion, and survival of Salmonella Typhimurium exposed to the simulated intestinal conditions.

This study was designed to evaluate the effect of bacteriophage P22 on the susceptibility, swimming motility, invasion gene expression, invasive ability, and intracellular survival of Salmonella Typhimurium exposed to the simulated intestinal conditions. S. Typhimurium cells were inoculated at 37 °C for 4 h in the simulated intestinal conditions with or without bacteriophage P22, including control (0 % bile salts, pH 7.2), SN (0 % bile salts, pH 5.0), SL (0.5 % bile salts, pH 5.0), SH (2.0 % bile salts, pH 5.0), SNp (0 % bile salts + P22, pH 5.0), SLp (0.5 % bile salts + P22, pH 5.0), and SHp (2.0 % bile salts + P22, pH 5.0). The numbers of Typhimurium cells were significantly reduced by 3.30, 3.56, and 3.75 log units, respectively, at SNp, SLp, and SHp. Considerable reduction in the swimming motility was observed at SNp (23 %), SLp (22 %), and SHp (20 %). The transcriptional regulator genes, hilA, hilC, hilD, invA, invE, and invF, were significantly down-regulated with SHp, showing 4.07-fold, 2.87-fold, 3.43-fold, 2.07-fold, 1.44-fold, and 4.83-fold, respectively. The decrease in invasive ability was most significant at SHp (45 %), followed by SLp (49 %). These results suggest that bacteriophage P22 can be used as an alternative to control Salmonella invasion of epithelial cells.